Generation of PAX7 Reporter Cells to Investigate Skeletal Myogenesis from Human Pluripotent Stem Cells

STAR Protoc. 2020 Nov 5;1(3):100158. doi: 10.1016/j.xpro.2020.100158. eCollection 2020 Dec 18.

Abstract

This protocol describes the use of CRISPR/Cas9-mediated homology-directed recombination to construct a PAX7-GFP reporter in human pluripotent stem cells (hPSCs). PAX7 is a key transcription factor and regulator of skeletal muscle stem/progenitor cells. We obtained heterozygous knockin reporter cells and validated their PAX7 expression using both artificial activation by the CRISPR/dCas9-VPR system and physiological activation during hPSC myogenic differentiation. These cells can serve as tools for better understanding of in vitro hPSC myogenesis and enriching myogenic cells for downstream analysis. For complete details on the use and execution of this protocol, please refer to Xi et al. (2017) and Xi et al. (2020).

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3' Untranslated Regions / genetics
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Binding Sites
  • CRISPR-Associated Protein 9 / metabolism
  • CRISPR-Cas Systems / genetics
  • Cell Count
  • Cell Differentiation
  • Conserved Sequence
  • Drug Resistance, Microbial
  • Genes, Reporter*
  • Genotype
  • Humans
  • Mammals
  • Mesoderm / embryology
  • MicroRNAs / genetics
  • MicroRNAs / metabolism
  • Muscle Development*
  • PAX7 Transcription Factor / chemistry
  • PAX7 Transcription Factor / metabolism*
  • Plasmids / genetics
  • Pluripotent Stem Cells / metabolism*
  • Protein Isoforms / chemistry
  • Protein Isoforms / metabolism
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • Reproducibility of Results
  • Somites / embryology

Substances

  • 3' Untranslated Regions
  • MicroRNAs
  • PAX7 Transcription Factor
  • Protein Isoforms
  • RNA, Guide, CRISPR-Cas Systems
  • CRISPR-Associated Protein 9